Preparation of novel derivatives of 2,3 dihydroxypropyl-n-(7 or 8 chloro-4 quinolinyl)anthranilate

ABSTRACT

A process of preparing novel derivatives of 2,3-dihydroxypropylN-(7 or 8-chloro-4-quinolinyl)anthranilate. The compounds are represented by the formula   WHEREIN THE CHLORO IS IN THE 7- OR 8-POSITION AND R is -CH2OCH3. The compounds are prepared by microbial transformation of 2,3dihydroxypropyl-N-(7 or 8-chloro-4-quinolinyl)anthranilate. The compounds are useful as analgesic and anti-inflammatory agents.

United States Patent 1 1 Theriault et al.

[ 1 Oct. 1, 1974 PREPARATION OF NOVEL DERIVATIVES OF 2,3DIHYDROXYPROPYL-N-(7 OR 8 CHLORO-4 QUINOLINYL)ANTI'IRANILATE [75]Inventors: Robert John Theriault, Kenosha,

Wis.; James Paul Karwowski, Mundelein; Norman Earl Wideburg, Waukegan,both of I11.

[73] Assignee: Abbott Laboratories, Chicago, Ill. [22] Filed: May 10,1973 [21] Appl. No.: 359,141

Related US. Application Data [62] Division of Ser. No. 190,690, Oct. 19,1971, Pat. No.

Primary ExaminerAlvin E. Tanenholtz Attorney, Agent, or Firm-Robert L.Niblack; Joyce R. Krei; Vincent A. Mallare ABSTRACT A process ofpreparing novel derivatives of 2,3-dihydroxypropyl-N-(7 or8-chloro-4-quinolinyl) anthranilate. The compounds are represented bythe formula IO o=oocm0HoHR wherein the chloro is in the 7- or'8-positionand R is CH OCH The compounds are prepared by microbial transformationof 2,3-dihydroxypropyl-N-( 7 or 8-chlor0-4-quinolinyl)anthranilate. Thecompounds are useful as analgesic and anti-inflammatory agents.

1 Claim, No Drawings 1 PREPARATION OF NOVEL DERIVATIVES OF 2,3

DIHYDROXYPROPYL-N-U on s CHLOR-4 QUINOLINYL)AN'I IIB ANILATE i This is adivision, of application Ser. No. 190,690 filed Oct. 19, 1971 now U.S.Pat. No. 3,790,578.

DETAILED DESCRIPTION OF THE INVENTION This invention relates to novelderivatives of 2,3-dihydroxypropyl-N-(7 or 8-chloro-4-quinolinyl)anthranilate and to a process for preparing the compounds viamicrobial transformation.

A number of humans and animals are known to suffer from variousrheumatic conditions involving inflammation, swelling, tenderness,decreased mobility, pain and fever. While there are presently availableantiinflammatory agents'which have been found to be effective in thesymptomatic treatment of such conditions as rheumatoid arthritis,rheumatoid spondylitis, osteoarthritis, gouty arthritis, and the like,such agents have a number of undesirable side effects. Thus, the searchfor improved anti-inflammatory agents continues.

The present invention provides novel compounds which are effectiveanti-inflammatory agents and further provides novel analgesic agents.

Accordingly, it is a primary object of this invention to provide novelderivatives of 2,3-dihydroxypropyl-N- (7 or8-chloro-4-quinolinyl)anthranilate.

A further object is to provide therapeutic composi tions for the reliefof inflammation and the accompanying pain, swelling, fever and the likein warm-blooded mammals.

Still a further object is to provide methods for treating inflammationand/or pain in mammals.

Further objects will become apparent to those skilled in the art fromthe following description and claims.

The compounds of this invention are represented by general Formula I N vI In addition to the analgesic activity, the compounds additionallyexhibit anti-inflammatory activity at oral dosages of from 5 to 300mg./kg. of body weight daily.

Therapeutic compositions comprising, as their active ingredient(s), oneor more compounds of Formula I, in

NH species U Bas s (F-55) NRRL- (IJ 3941 I O: OCHgOHOHCHzOH vb v. a...01

wherein the chloro is thesis of the compounds of Formula II,2,3-dihydroxypropyl-N-( 7- chloro-4-quinolinyl)anthranilate and 2,3-

dihydroxypropyl-N-( 8-chloro-4- quinolinyl)anthranilate, are describedin Belgian Patent Numbers 678,551, published Sept. 28, 1966 (8- chloro)and 636,381, published Feb. 20, 1964 (7- chloro). The compounds ofFormula II also exhibit anti-inflammatory and analgesic activity.

The following fermentation media are used in the practice of thisinvention:

Fermentation Media A Grams/liter Glucose monohydrate (added poststerilization) 50.0 Soy fluff flour 5.0 Yeast extract 5.0 KH PO 2.3 KHPO 0.84

The pH is adjusted to approximately 6.5 and deionized water is added toadjust the volume to 1.0 liter.

The following examples further illustrate this invention.

Example 1 Preparation of 2-Hydroxy-3-Methoxypropyl-N-(7-Chloro-4-Quinolinyl)Anthranilate Basidiomycete species U Bas-8 (F-55)NRRL-394l was inoculated from 14 day incubated agar slant cultures intoa series of sterile cotton plugged 500 ml. Erlenmeyer flasks eachcontaining ml. of fermentation medium A. The inoculated flasks wereincubated on a Gump rotary shaker (250 rpms) at 28C. for 48 hours to 72hours. At that time, 0.050.l percent of 2,- 3-dihydroxypropyl-N-(7-chloro-4-quinolinyl C OCH CHOHR in the 7 or 8-position. Thesynanthranilate or 0.05-0.1

dihydroxypropyl-N-(8-chloro-4- quinolinyl)anthranilate (50 mg./100 ml.medium) was added in powdered form. The flasks were again incubated onthe shaker and were sampled at various ages during the fermentation forthin-layer chromatography (TLC) analysis in order to determine theoptimum harvest age as follows: ml. of whole culture were adjusted to pH9.0 with NH OH. One volume of, acetone was added with mixing and thesamples were then extracted with 2 volumes of ethyl acetate, twice. Theethyl acetate extracts were dried in vacuo and the residue obtained wasreconstituted in 2 ml. of ethanol. The ethanol solutions were spotted(200 microliters) on cm X 20 cm glass plates coated with Merck-Darmstadtsilica gel GF about 500 microns in thickness. The thin-layerchromatography (TLC) plates were developed in a solvent systemconsisting of CHCl 3:CH OH:NH OH (85:15: 1 The substrate and microbialconversion product were revealed on TLC plates as dark UV absorbingspots with 254 nm UV light and orange to brown spots after the plateswere sprayed with Dragendorffs reagent. Thin-layer chromatography R s ofthe substrate and resulting microbial conpercent of 2,3-

chloro-4-Quinolinyl)Anthranilate Basidiomycete species F-55, NRRL-394 l,was inoculated at a level of one 14 day agar slant culture suspensionper flask to approximately 200 sterile, cottonplugged 500 ml. Erlenmeyerflasks each containing 100 ml. of medium A. The flasks were incubated ona Gump rotary shaker (250 rpm at 28C. for 72 hours, at which time 0.05percent (50 mg./l00 ml. of medium) of 2,3-dihydroxypropyl-N-(7-chloro-4-quinolinyl)anthranilate was added to each flask in powdered form. Theflasks were again incubated on the shaker and were sampled at variousages, as described in Example 1, for thin-layer chromatography analysisin order to determine the optimal age for harvest. When the optimalyield of the microbial conversion product was reached, all flasks wereharvested and the whole culture from each flask was pooled and adjustedto pH 2. The whole culture was filtered and the mycelium discarded afterwashing the pH 2 water. The filtered fermentation beer was concentratedto one-fifth volume under vacuum at C. and then adjusted to pH 9 with NHOH. The concentrated pH 9 beer was then extracted once with 2 volumes ofethyl acetate. The ethyl acetate extract was dried under vacuum andredissolved in 23A ethyl alcohol: H O(75:25). This solution was filteredto remove any precipitate and the clear solution was extacted with 3volumes of CCh. The CCl, extract was dried and chromatographed on aLH-20 Sephadex column packed by the slurry method and developed with asolvent solution consisting of heptanezCHCl z 23A ethyl alcohol (10:82).The major microbial conversion product, 2-hydroxy-3-methoxy-N-(7-chloro-4-quinolinyl )anthranilate, mp. 17 1 73, was crystallizeddirectly from column eluate fractions. The 100 MHz nmr spectrum wasconsistent with the following structure:

The mass spectrum confirmed this structure and gave a molecular ion (M)of 386.1012 (C H N O Cl) Analysis Calcd. for C,.,H N O Cl: C, 62.09; H,4.95; N. 7.24;

Found: C, 61.91; H, 4.97; N, 7.19;

While the compound can be administered alone, that is, as the solecomponent in a filled capsule, it is preferred to formulate the compoundin various dosage forms for oral administration such as tablets, syrupsand the like. Such dosage forms are prepared by methods well known inthe art and generally include a pharmaceutically acceptable carrier ordiluent such as lactose, starch or sucrose along with lubricating agentssuch as magnesium stearate and flavoring and sweetening agents and thelike.

What is claimed is:

1. A process of preparing a compound wherein the chloro is in the 7- orS-position and R is CH OCH said process comprising culturingBasidomycete species NRRL-394l in a nutrient medium containing suitablesources of carbon, nitrogen and minerals in the presence of a secondcompound of the formula iOCHgCHOHCH OH wherein the chloro is in the 7-or 8-position for a sufficient length of time to transfonn said secondcompound to said desired compound.

1. A PROCESS OF PREPARING A COMPOUND